Pharmaceutical composition for prevention and/or treatment of bone loss

ABSTRACT

A pharmaceutical composition for preventing and/or treating bone loss is disclosed. The pharmaceutical composition includes an effective amount of a licorice, black bean, Cnidi Fructus, and buckhorn.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending application Ser. No.12/044,577, filed on Mar. 7, 2008, and for which priority is claimedunder 35 U.S.C. §120; the entire contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a pharmaceutical composition, and inparticular relates to a pharmaceutical composition for preventing ortreating bone loss.

2. Description of the Related Art

Osteoporosis patients have been divided into two groups includingpostmenopausal osteoporosis and senile osteoporosis. Postmenopausalosteoporosis generally occurs in older aged women and is caused byfemale hormone deficiency. Senile osteoporosis generally occurs in olderaged men and women. Human bone consists of a hard bone cortex and a softmedullary cavity that contains bone marrow, and the ratio of bone cortexand bone marrow has significant variations at different positions.Postmenopausal osteoporosis usually results in bones mainly consistingof bone marrow, thus leading to fractures, such as lumbar compressionfractures and wrist fractures.

Postmenopausal osteoporosis is a metabolic bone disease suffered byolder aged women. In clinical research, bone loss reaches up to 3˜5%annually for cases with hyposecretion of estrogen for climacteric women.Due to the prolonged lifespan of humans, the incidence trend ofpostmenopausal osteoporosis has been rising. Additionally,postmenopausal osteoporosis is primarily caused by numerous bone lossdue to the deficiency of estrogen (approx. 3-6% or higher every year).In normal state, the excited hormone can stimulate osteoblast togenerate cells, which depress the activity of the osteoclast, andstimulate osteoblast to keep the bone in a balanced state. In theabsence of estrogen, numerous bones will be lost and calcium ion will bereleased from the bone, thus inhibiting the concentration of parathyroidhormone and leading to reduced composition of vital vitamins. This willreduce the calcium ion absorbed by the gastrointestinal tract, leadingto a calcium ion imbalance, which adds to the rising trend ofosteoporosis.

Although postmenopausal osteoporosis may be suppressed by hormonetreatment, use of large amounts of hormone over a long period of timemay cause serious side effects (Colditz G A, Hankinson S E, Hunter D J.The use of estrogen and progestins and the risk of breast cancer inpostmenopausal women. N Eng J Med, 1995, 332:1589-1593; Grady D,Gebretsadik T, Kelikowske K. Hormone replacement therapy and endometrialcancer risk: a meta-analysis. Obstet Gynecol, 1995, 85:304-313). Apharmaceutical composition and method for treating or suppressing boneloss are thus required.

BRIEF SUMMARY OF INVENTION

The present invention provides a method for preventing, ameliorating,and/or treating bone loss, comprising administering to a subject aneffective amount of a pharmaceutical composition comprising licorice,black bean, Cnidi Fructus, and buckhorn, wherein a weight ratio oflicorice, black bean, Cnidi Fructus, and buckhorn is about 1-10:2-10:1-10:1-10.

The present invention further provides a method for preventing,ameliorating, and/or treating bone loss, comprising administering to asubject an effective amount of a pharmaceutical composition comprisingimperatorin and a pharmaceutically acceptable carrier or excipient.

The present invention further provides a method for preventing,ameliorating, and/or treating bone loss, comprising administering to asubject an effective amount of a pharmaceutical composition comprisingbergapten and a pharmaceutically acceptable carrier or excipient.

A detailed description is given in the following embodiments withreference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

The present invention can be more fully understood by reading thesubsequent detailed description and examples with references made to theaccompanying drawings, wherein:

FIG. 1 shows the effect of the pharmaceutical composition of theinvention on body weight of rats;

FIG. 2 is a photomicrograph showing the bone tissue of the rats treatedwith the pharmaceutical composition of the invention;

FIGS. 3A-3B shows the effect of the pharmaceutical composition of theinvention on cell proliferation;

FIG. 4 shows the effect of the pharmaceutical composition of theinvention on ALP activity in the bone cells;

FIGS. 5A-5B shows the effect of the pharmaceutical composition of theinvention on bone mineralization;

FIG. 6A show the effect of the pharmaceutical composition of theinvention on bone cell differentiation;

FIG. 6B shows the effect of the pharmaceutical composition of theinvention on bone resorption;

FIG. 7A shows the effect of imperatorin and bergapten on cell viability;

FIG. 7B shows the effect of imperatorin and bergapten on cellproliferation;

FIG. 8A shows the effect of imperatorin and bergapten on ALP activity inthe bone cells;

FIG. 8B shows the effect of imperatorin and bergapten on collagensecretion in the bone cells;

FIGS. 8C-8D show the effect of imperatorin and bergapten on bonemineralization;

FIGS. 9A-9B show the effect of imperatorin and bergapten on BMP-2 geneexpression;

FIG. 9C shows the effect of imperatorin, bergapten, and noggin on BMP-2gene expression;

FIGS. 10A-10B shows the effect of imperatorin and bergapten onphosphorylation of SMADs, p38, and EPK protein in the bone cells;

FIGS. 11A-11B show the effect of p38 and ERK inhibitor onphosphorylation of p38 and EPK protein in the bone cells;

FIG. 11C shows the effect of p38 and ERK inhibitor on ALP activity inthe bone cells;

FIG. 11D shows of imperatorin and bergapten on ALP activity in p38mutant cell (DN-p38) and ERK mutant cell (DN-ERK), and

FIG. 12 is a photomicrograph showing the increase of the bone volume intibia metaphysic by imperatorin and berapten.

DETAILED DESCRIPTION OF INVENTION

The following description is of the best-contemplated mode of carryingout the invention. This description is made for the purpose ofillustrating the general principles of the invention and should not betaken in a limiting sense. The scope of the invention is best determinedby reference to the appended claims.

The present invention provides a pharmaceutical composition forpreventing, ameliorating and/or treating bone loss, comprising aneffective amount of licorice, black bean, Cnidi Fructus, and buckhorn,wherein a weight ratio of licorice, black bean, Cnidi Fructus, andbuckhorn is about 1-10:2-10:1-10:1-10, preferably, about2-4:4-6:1-3:1-3. The pharmaceutical composition also contains osthol,imperatorin, and/or bergapten.

The pharmaceutical composition of the invention can be manufacturedusing a method as follows. Firstly, licorice is heated and extractedwith an extract solution to obtain a licorice extract, wherein theextract solution includes, but is not limited to, water or ethanol.Block bean and Cnidi Fructus are soaked in ethanol for 2 to 40 hours,preferably, 10 to 20 hours, and then extracted using high-temperatureheat. Subsequently, the extracted licorice, Block bean, and CnidiFructus are combined, and the volume ratio of licorice and Blockbean+Cnidi Fructus is 1:2. The combined extract is concentrated underreduced pressure to give a concentrate (10 to 100 liter). Theconcentrate is mixed with 10 to 40 kg of buckhorn powder, dried, andformed to obtain the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention can treat, prevent,reduce, or ameliorate bone loss including osteoporosis or anosteoporotic fracture. All bone mineral density (BMD), bone mineralcontent (BMC), and alkaline phosphatase (ALP) activity of a subject areincreased by the pharmaceutical composition. The BMD, BMC, and ALPactivity can be increased by at least 5.15%, 3.77%, and 30.0%,preferably, 10.30%, 11.32%, and 90.0%, respectively. Additionally,maximal load, ultimate loading, Young's modulus, and ultimate stress ofa bone tissue in a subject also are increased by the pharmaceuticalcomposition. The maximal load, ultimate loading, Young's modulus, andultimate stress can be increased by at least 3.14%, 5.23%, 7.49%, and15.65%, preferably, 10.24%, 13.2%, 10.1%, and 34.8%, respectively.

In one embodiment, the pharmaceutical composition comprises an effectiveamount of imperatorin, and a pharmaceutically acceptable carrier orexcipient. The composition may be administered orally or by injection.Following the treatment with the composition containing imperatorin,bone mineral density, bone mineral content, and alkaline phosphatase(ALP) activity of a subject can be increased by at least 12.22%, 28.91%,and 6.0%, respectively. Preferably, the alkaline phosphatase (ALP)activity can be increased by at least 95%. Additionally, the collagencontent and mineralization can also be increased by at least 102.0% and110.0%, respectively.

In another embodiment, the pharmaceutical composition comprises aneffective amount of bergapten, and a pharmaceutically acceptable carrieror excipient. The composition may be administered orally or byinjection. Following the treatment of the composition containingbergapten, bone mineral density, bone mineral content, and alkalinephosphatase (ALP), activity of a subject can be increased by at least12.2%, 33.7%, and 80.0%, respectively, and the collagen synthesis andmineralization can be increased by at least 100%, and 105.0%,respectively.

The term “subject”, as used herein, means an animal, including a humanor non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep,a pig, a goat, or a non-human primate, and expressly includes laboratorymammals, livestock, and domestic mammals. In one embodiment, the mammalmay be a human, such as an osteoporosis patient; in another, the mammalmay be a rodent, such as a mouse or a rat.

The pharmaceutical composition of the invention may be administered byany suitable means, including, without limitation, parenteral,intravenous, intramuscular, subcutaneous, implantation, oral,sublingual, buccal, nasal, pulmonary, transdermal, topical, vaginal,rectal, and transmucosal administrations or the like. Topicaladministration can also involve the use of transdermal administrationsuch as transdermal patches or iontophoresis devices. Pharmaceuticalpreparations include a solid, semisolid or liquid preparation (tablet,pellet, troche, capsule, suppository, cream, ointment, aerosol, powder,liquid, emulsion, suspension, syrup, injection etc.) containing acompound of the invention as an active ingredient, which is suitable forthe selected mode of administration. In one embodiment, thepharmaceutical compositions are administered orally, and are thusformulated in a form suitable for oral administration, i.e., as a solidor a liquid preparation. Suitable solid oral formulations includetablets, capsules, pills, granules, pellets, sachets and effervescent,powders, and the like. Suitable liquid oral formulations includesolutions, suspensions, dispersions, emulsions, oils and the like.

EXAMPLE Example 1 Manufacturing of the Pharmaceutical Composition of theInvention

The pharmaceutical composition was made up as follows: 30 kg oflicorice, 60 kg of black bean, 10 kg of Cnidi Fructus, and 8 kg ofbuckhorn. The licorice was heated to 100° C., and then extracted withwater to give an extract (150 liter). Black bean and Cnidi Fructus weresoaked in 30% ethanol for 20 hr at 80° C., and combined with the extractof licorice, wherein the volume ratio of licorice and Block bean+CnidiFructus was 1:2. The combined extract was concentrated under reducedpressure to give a concentrate (30 liter). The concentrate was mixedwith buckhorn powder, dried at 60° C. for 48 hours, and formed to obtainthe pharmaceutical composition of the invention.

Example 2 Preparation of Bone Tissue

Eight week old female Sprague-Dawley (SD) rats were purchased from theanimal center of the National Laboratory Animal Breeding and ResearchCenter of the National Science Commission. Each rat was caged alone andallowed feedstuff and drinking water for 4 weeks before experimentation.The rats were classified into three groups: (1) sham group, (2)ovariectomy (OVX) group, and (3) OVX+pharmaceutical composition of theinvention group. In groups (2) and (3), the rats were anesthetized withtrichloroacetaldehyde (400 mg/kg) in a sterile operating environment,whereby access was given to the back of the rat's abdominal cavity fromthe split lumbar, and the ovaries of both sides of the rat's were fullyremoved. No ovariectomy was required for group (1) (sham group) withonly surgical stitching. The feedstuff and drinking water were takensimilarly as before. One day after ovariectomy, the rats were treatedwith the pharmaceutical composition of the invention (1.2 g/kg) by oraladministration everyday in group (3). In groups (1) and (2), the ratswere treated with carboxymethyl cellulose. The dosage was modifiedweekly for six weeks according to the body weight, and then all ratswere slaughtered.

Example 3 Measurement of Bone Length and Weight

Rat femur and tibia were isolated from the rats after being slaughteredand kept at −80° C. The muscles and connective tissues were removed fromthe femurs and tibias. Then, the bone weight was measured by amicroblancer, and bone length was measured by a micro scale (±0.05 mm).Bone mineral density (BMD) and content (BMC) were measured using adual-energy X-ray absorptiometer (DEXA, XR-26; Norland, Fort Atkinson,Wis.). Additionally, the femurs were fixed in 4% formalin for two days,and then treated with 10% EDTA at 4° C. for two weeks ofdecalcification. After the treatment, the femurs were dehydrated byalcohol, buried into olefin for 5 mm slicing, and stained with Mayer'shematoxylin-eosin. The bone volume of secondary spongiosa was measuredby using an image-pro plus (3.0 ed). Referring to FIG. 1, the boneweight of the OVX group rats were heavier than that of the sham grouprats, and the pharmaceutical composition did not cause a decrease inbody weight of the rats. Referring to FIG. 2, the tissue sectionsdemonstrated that the pharmaceutical composition of the inventionsuppressed bone loss. The experimental results are listed in Table 1.

TABLE 1 OVX + I OVX + I OVX + I Sham OVX (1.2 g/kg) (0.8 g/kg) (0.4g/kg) (n = 26) (n = 26) (n = 14) (n = 13) (n = 13) Bone length (mm)Tibia 4.03 ± 0.02 4.02 ± 0.02  4.04 ± 0.03  3.99 ± 0.02 4.03 ± 0.0.1 Femur 3.62 ± 0.04 3.65 ± 0.05  3.67 ± 0.0.5  3.62 ± 0.04 3.62 ± 0.03 Wet weight (mg) Tibia  758 ± 10.5  657 ± 12.7*  722 ± 10.6^(#) 716.8 ±7.4^(# )  693.3 ± 17.1^(#)  Femur 958.1 ± 10.3   862 ± 13.9* 911.5 ±9.6^(#)    923 ± 25.4^(#)  895 ± 15.5^(#) BMD (g/cm3) Tibia 0.109 ±0.009 0.097 ± 0.011* 0.107 ± 0.01^(#)  0.102 ± 0.006 0.105 ± 0.006 Femur 0.131 ± 0.007 0.122 ± 0.008* 0.127 ± 0.011^(#)  0.128 ± 0.028^(#)0.127 ± 0.016^(#) BMC (g) Tibia 0.299 ± 0.012 0.265 ± 0.014* 0.295 ±0.024^(#)  0.290 ± 0.002^(#) 0.275 ± 0.003^(#) Femur 0.411 ± 0.014 0.357± 0.013* 0.402 ± 0.022^(#)  0.415 ± 0.025^(#) 0.408 ± 0.014^(#) Bone17.8 ± 1.6  9.3 ± 1.5* 15.7 ± 1.5^(#)  14.9 ± 1.9^(# ) 13.4 ± 2.5^(#) volume (%) OVX: ovariectomy I: the pharmaceutical composition of theinvention *p < 0.05, compared with the sham group ^(#)p < 0.05, comparedwith the OVX group

Example 4 Biomechanics Analysis of Bone Tissue

Biomechanics analysis was dependent upon the measurement of athree-point test by a material testing system (MTS-858, MTS System Inc.,Minneapolis, Minn.). The distance from both sides of a bone was 20 mm,and filling speed was 1 mm/m. Young's modulus was obtained by acquiringthe regression line of the speed and pressure and substituting both intothe following formulas. σ=FLc/41, E=F/d′L/481. Wherein, σ is ultimatestress, c is the distance from the center of mass (equal to ½b asdescribed above), F is the applied load (N), d is the displacement (mm),and L is the span between the two support points of the bending fixture(mm).

TABLE 2 OVX + I OVX + I OVX + I Sham OVX (1.2 g/kg) (0.8 g/kg) (0.4g/kg) (n = 26) (n = 26) (n = 14) (n = 13) (n = 13) Maximal 102.7 ± 2.985.9 ± 3.0* 94.7 ± 2.8^(#) 91.7 ± 2.9^(#) 88.6 ± 1.5 load (N) Ultimate  81 ± 4.6 70.7 ± 3.8*  80 ± 2.4^(#) 76.1 ± 2.3^(#) 74.4 ± 1.8 loading(N) Young's  201 ± 6.2 170.8 ± 6.6*   188 ± 6.8^(#) 187.5 ± 5.9^(#) 183.6 ± 4.2^(# ) modulus (GPa) Ultimate  18.1 ± 0.9 11.5 ± 1.5* 15.5 ±1.0^(#) 15.8 ± 1.2^(#)  13.3 ± 1.4^(#) stress (MPa) OVX: ovariectomy I:the pharmaceutical composition of the invention *p < 0.05: compared withthe sham group ^(#)p < 0.05: compared with the OVX group

Example 5 Culture of Rat Osteoblast

Rat primary osteoblast was isolated from the parietal bone of gestationday 18 embryos of Spraque-Dawley rats. After being slaughtered, theembryo rats were obtained from the uteruses of the rats, and calvariumwas isolated from the embryo rats. Parietal bone was cut from thecalvarium and digested by 0.1% collagenase for 20 minutes to release thebone cells from the bone tissue. The digestion step was repeated fivetimes. The collected bone cells were cultured in α-MEM containing 1%penicillin-streptomycin and 10% FBS in a humidified atmosphere of 5percent carbon dioxide. When cells were attached and confluence, theywere sub-cultured, which was called “passage 2 (P2)”. The passage wasfor analogizing. 2 to 5 passage cells were used for the experiments.

Example 6 Effect of Each Component on Cell Proliferation

Rat osteoblast cells were isolated and cultured in 96-well Plateaccording to the methods described above. The osteoblase cells weretreated with 5, 15, 50, and 100 μg/ml of (1) licorice, (2) blackbean+Cnidi Fructus, (3) Cnidi Fructus, (4) black bean, and (5) thepharmaceutical of the invention for 48 hrs, respectively and then themedium was removed. Proliferation of the osteoblast cells was testedusing Cell Proliferation ELISA, BrrdU kit (Roche Applied Science). Theproliferation level in the control group which was treated with 0.1%DMSO was defined as 100%. Referring to FIGS. 3A-3B, when compared withthe control group, all components increased the proliferation of theosteoblast cells.

Example 7 Effect of Each Component on Alkaline Phosphatase (ALP)Activity

Rat osteoblast cells were isolated and cultured in 96-well Plateaccording to the methods described above. The osteoblast cells weretreated with 5, 15, 50, and 100 μg/ml of (1) licorice, (2) blackbean+Cnidi Fructus, (3) Cnidi Fructus, (4) black bean, and thepharmaceutical of the invention extract, respectively. The treatedosteoblast cells were digested in 0.2% NP-40 solution, and thencentrifuged at 1500×g for 5 minutes to obtain a supernatant. Thesupernatant was analyzed to obtain the ALP activity of the osteoblastcells using an ALP kit (Roche Applied Science). The ALP activity levelin the control group which was treated with 0.1% DMSO was defined as100%. Referring to FIG. 4, all treatments except those treated by theblack bean increased the ALP activity of the osteoblast cells.

Example 8 Effect of Each Component on Bone Mineralization

Rat osteoblast cells were cultured in 96-well Plate containing adifferentiation medium (5 μg/ml vitamin C and 10 mM β-glycerophosphate)and treated with 5, 15, 50, and 100 μg/ml of (1) licorice, (2) blackbean+Cnidi Fructus, (3) Cnidi Fructus, (4) black bean, and (5) thepharmaceutical of the invention, respectively. The differentiationmedium was refreshed every 3 days. 14 days after the cell culture, theosteoblast cells were fixed with 75% ethanol for 30 minutes, and thentreated with 40 mM Alizarin red-S at room temperature for 1 hr. Afterthe addition of Alizarin red-S, 10% cetylpyridinium chloride was added,and then absorbance was measured at 550 mm. The mineralization level inthe control group which was treated with 0.1% DMSO was defined as 100%.Referring to FIGS. 5A-5B, all treatments except those treated by blackbean increased the mineralization of the osteoblast cells.

Example 9 Effect of Each Component on Differentiation of the OsteoclastCells

Rat osteoclast cells were isolated from the bone marrow of SpragueDawley (SD) rats by a needle. 1×10⁶ osteoclast cells were cultured in aplate with a medium containing a differentiation-inducing agent (50ng/ml PANKL and 20 ng/ml M-CSF), wherein the differentiation-inducingagent was refreshed every 3 days. The cells were treated with 150 μg/mlof (1) licorice, (2) black bean+Cnidi Fructus, (3) Cnidi Fructus, (4)black bean, and (5) pharmaceutical of the invention extract,respectively. Eight days after the cell culture, the cells were stainedwith tartrate-resistant acid phosphatats (TRAP), and then theTRAP-stained cells (TRAP-positive cell) were measured using amicroscope. In the control group, the osteoclast cells were treated with0.1% DMSO. Referring to FIG. 6A, the differentiation of the osteoclastcells was only slightly suppressed by black bean.

Example 10 Effect of Each Component on Resorption of the OsteoclastCells

Rabbit osteoclast cells were isolated from the long bones of New ZealandWhite Rabbits (age 5 days) and cultured in OAAS plate. The osteoclastcells were treated with 150 μg/ml of (1) licorice, (2) black bean+CnidiFructus, (3) Cnidi Fructus, (4) black bean, and (5) pharmaceutical ofthe invention extract, respectively. Three days after treatment, thecells were digested with 1 N NaOH, and then washed three times.Following the wash, the cells were photographed under a microscope andanalyzed to obtain the resorption area of the osteoclast cells using theImage-Pro Plus 3.0 software. In the control group, the osteoclast cellswere treated with 0.1% DMSO. Referring to FIG. 6B, the resorption of theosteoclast cells was not suppressed by all components.

Example 11 Effect of Imperatorin and Bergapten on Cell Viability

Bone cells were cultured in 96-well plate and treated with 0.3, 1, 3,and 10 μM of imperatorin and bergapten for 48 hrs, respectively. Aftertreatment, the cells were reacted with MIT solution (0.5 mg/ml) at 37°C. for 30 minutes, lysed in 100 μl DMSO, and then absorbance wasmeasured at 550 mm. The cell viability in the control group which wastreated with 0.1% DMSO was defined as 100%. Referring to FIG. 7A, bothimperatorin and bergapten did not suppress the cell viability.

Example 12 Effect of Imperatorin and Bergapten on Proliferation of BoneCells

Bone cells were cultured in 96-well Plate and treated with 0.3, 1, 3,and 10 μM of imperatorin and bergapten. After treatment, the medium wasremoved. The proliferation of the bone cells was tested using CellProliferation ELISA, BrrdU kit (Roche Applied Science). Theproliferation level in the control group which was treated with 0.1%DMSO was defined as 100%. Referring to FIG. 7B, both imperatorin andbergapten were not suppressed the proliferation of the bone cells.

Example 13 Effect of Imperatorin and Bergapten on ALP Activity of BoneCells

Bone cells were treated with 0.3, 1, 3, and 10 μM of imperatorin andbergapten, respectively. After treatment, the osteoblast cells weredigested in 0.2% NP-40 solution, and then centrifuged at 1500 g for 5minutes. The ALP activity of the osteoblast cells was tested using anALP kit (Roche Applied Science). The ALP activity level in the controlgroup which was treated with 10% DMSO was defined as 100%. Referring toFIG. 8A, imperatorin and bergapten both increased the ALP activity ofthe bone cells.

Example 14 Effect of Imperatorin and Bergapten on Collagen Secretion inthe Bone Cells

Bone cells were treated with 0.3, 1, 3, and 10 μM of imperatorin andbergapten, respectively. After treatment, the cells were lysed in 6N HClat 116° C. for 16 hrs, and dried in a vacuum. The resultant residue wasdissolved in an equal volume of water, and then absorbance was measuredat 550 mm. The collagen secretion in the control group which was treatedwith 0.1% DMSO was defined as 100%. Referring to FIG. 8B, imperatorinand bergapten both increased the collagen secretion in the bone cells

Example 15 Effect of Imperatorin and Bergapten on the Mineralization inthe Bone Cells

Bone cells were cultured in 96-well Plate containing a differentiationmedium (50 μg/ml vitamin C and 10 mM β-glycerophosphate) and treatedwith 0.3, 1, 3, and 10 μM of imperatorin and bergapten, respectively.The differentiation medium was refreshed every 3 days. 14 days after thecell culture, the bone cells were fixed with 75% ethanol for 30 minutes,and then treated with 40 mM Alizarin red-S at room temperature for 1 hr.After the addition of Alizarin red-S, 10% cetylpyridinium chloride wasadded, and then absorbance was measured at 550 mm. The mineralizationlevel in the control group which was treated with 0.1% DMSO was definedas 100%. Referring to FIGS. 8C-8D, imperatorin and bergapten bothincreased the mineralization of bone cells.

Example 16 Effect of Imperatorin and Bergapten on the Expression of theBMP-2 Gene

Bone cells were cultured in 6-well Plate and cultured with 0.3, 1, 3,and 10 μM of imperatorin and bergapten for 1, 3, 6, and 12 hrs,respectively. After treatment, the cells were lysed with TRIzol reagentat room temperature for 5 minutes, and then collected in a centrifugetube. 0.5 ml of chloroform was added to the centrifuge tube. After theaddition of chloroform, the centrifuge tube was well mixed, andcentrifuged at 14000×g for 15 minutes at room temperature to obtain asupernatant. The supernatant was mixed with isopropanol (0.25 ml) andthen centrifuged at 14000×g at room temperature for 15 minutes to obtaina precipitated RNA. RNA was washed with 75% alcohol, centrifuged at14000×g for 10 minutes, and dried to give a RNA pellet. The RNA pelletwas dissolved in diethyl pyrocarbonate (DEPC) solution and thenquantitated by measurement of OD_(260/280). BMP-2 gene expression (BMP-2mRNA level) was detected by RT-PCR using Supperscript™ IIIreverstranscriptase and ABI Primer 700 (Applied Biosystems). The BMP-2mRNA level in the control group which was treated with 0.1% DMSO wasdefined as 100%. Referring to FIG. 9A, BMP-2 mRNA level was increasedaccording to the increase of the concentration of imperatorin orbergapten. Referring to FIG. 9B, BMP-2 mRNA level was increasedaccording to the increase of the treatment time.

Example 17 Effect of Imperatorin, Bergapten and Noggin on the Expressionof the BMP-2 Gene

Bone cells were treated with (1) 10 μM imperatorin, (2) 10 μM bergapten,(3) 10 μM imperatorin+noggin, (4) 10 μM bergapten+noggin, and (5) 1μg/ml noggin for 48 hrs, respectively. After treatment, the bone cellswere lysed in 0.2% NT-40 solution, and then centrifuged at 1500×g for 5minutes to obtain a supernatant. The supernatant was analyzed by ALP kitas described above to obtain the ALP activity. The ALP activity in thecontrol group which was treated with 0.1% DMSO was defined as 100%.Referring to FIG. 9C, noggin suppressed the imperatorin or bergapteninduced-ALP activity. Thus, it indicated that imperatorin or bergaptenmay increase BMP-2 expression to induce bone cells differentiation.

Example 18 Effect of Imperatorin and Bergapten on Phosphorylation ofSMADs, p38, and EPK Protein in the Bone Cells

Bone cells was cultured in 6-well plate and treated with 10 μM ofimperatorin and bergapten for 1, 3, 6, 12, and 24 hrs, respectively.After treatment, the culture medium was replaced with a lysis buffercontaining 50 mM HEPES (PH 7.4), 150 mM NaCl, 4 mM EDTA, 10 mM Na₄P₂O₇,100 mM NaF, 2 mM Na₃VO₄, 1% (v/v) Triton X-100, 0.25% (w/v) sodiumdeoxycholate, 50 mM 4-(2-aminoethyl) benzene sulfonylfluoride, 50 μg/mlleupeptin, and 20 μg/ml aprotinin, and then centrifuged at 13000 rpm for15 minutes to obtain the protein pellets. 30 μg of proteins weredissolved in 5× Laemmli buffer, boiled at 95° C. for 5 minutes, and thensubjected to SDS-PAGE gel (8%) electrophoresis. Proteins on the gel werethen transferred to a PVDF membrane. The PVDF membrane was blocked in 4%BSA solution for 1 hour at room temperature. The blocked membrane wasthen incubated in PBST buffer (0.1% Tween-20) containing p-SMAD, p-ERK,or p38 primary antibody. Following incubation with primary antibody, themembrane was washed three times with PBST, and then incubated for 1 hrat room temperature with the secondary antibody, an anti-mouse oranti-goat IgG labeled with horseradish peroxidase. Following incubationwith the secondary antibody, the membrane was washed three times withPBST, and detected by an enzyme-linked chemiluminescence using an ECLblotting substrate according to the manufacturer's instructions.Referring to FIG. 10, the phosphorylation of SMADs, p38, and EPK proteinwere induced by imperatorin and bergapten.

Example 19 Effect of p38 Inhibitor (SB203580) and ERK Inhibitor(PD98059) on Phosphorylation of p38 and EPK Protein

The same procedure carried out in Example 18 was repeated except thatthe bone cells were changed to be treated with p38 inhibitor (SB203580)and ERK inhibitor (PD98059) for 30 minutes, before the treatment ofimperatorin and bergapten. In the control group, a non-phosphorylationp38 and non-phosphorylation EPK proteins were subjected to SDS-PAGE gelelectrophoresis. Referring to FIGS. 11A-11B, p38 inhibitor (SB203580)and ERK inhibitor (PD98059) suppressed the imperatorin and bergapteninduced-phosphorylation.

Example 20 Effect of p38 Inhibitor (SB203580) and ERK Inhibitor(PD98059) on ALP Activity in the Bone Cells

Bone cells were cultured and treated with (1) 10 μM SB203580, (2) 10 μMPD98059, (3) 10 μM imperatori, (4) 10 μM bergapten, (5) 10 μMimperatorin+SB203580, (6) 10 μM bergapten+SB203580, (7) 10 μMimperatorin+PD98059

(8) 10 μM bergapten+PD98059 for 48 hrs. After treatment, the bone cellswere lysed in 0.2% NT-40 solution and centrifuged at 1500×g for 5minutes. The supernatant was analyzed by an ALP kit such as describedabove to obtain the ALP activity. The ALP activity in the control groupwhich was treated with 0.1% DMSO was defined as 100%. Referring to FIG.11C, p38 inhibitor (SB203580) and ERK inhibitor (PD98059) suppressed theimperatorin and bergapten induced-ALP activity.

Example 21 Effect of Imperatorin and Bergapten on ALP Activity in p38Mutant Cell (DN-p38) and ERK Mutant Cell (DN-ERK)

The same procedure carried out in Example 20 was repeated. Theexperiment group used alternative cells and treatment comprising: (1)DN-p38 cell, (2) DN-ERK cell, (3) DN-p38 cell+10 μM imperatorin, (4)DN-p38 cell+10 μM bergapten, (5) DN-ERK cell+10 μM imperatorin, (6)DN-ERK cell+10 μM bergapten, (7) normal bone cell+10 μM imperatorin, and(8) normal bone cell+10 μM bergapten. The ALP activity in the controlgroup which was treated with 0.1% DMSO was defined as 100%. Referring toFIG. 11D, the ALP activity was not induced in p38 mutant cell (DN-p38)and ERK mutant cell (DN-ERK) by imperatorin and bergapten. Thus, theresults indicate that the induction of the ALP activity relates toSMADs, p38, and EPK proteins.

Example 22 Animal Experiment Male Spraue-Dawley rats (weighing 78 to 90grams, at three weeks of age) were anesthetized with 400 mg/ml oftrichloroacetaldehyde monohydrate. A 22G needle was sterilized andinjected into the tibia of the rats. One day after the injection, therats were administered with normal saline as the control, 30 μMimperatorin, and 30 μM bergapten every day for a week through theneedle, respectively. Then, the rats were slaughtered after the lastadministration, and the tibias were immediately isolated afterslaughtering and subject to BMD, BMC, and histochemical examination.Referring to FIG. 12, the bone volume of tibia metaphysis was increasedby imperatorin and bergapten. The results of the BMD and BMCexaminations are shown in Table 3.

TABLE 3 Control Imperatorin Bergapten BMD (g/cm³)  0.09 ± 0.001 0.101 ±0.002* 0.101 ± 0.002* BMC (g) 0.083 ± 0.002 0.107 ± 0.003* 0.111 ±0.003* BV/TV (%) 8.63 ± 0.2  17.6 ± 0.6*  18.1 ± 0.4*  BV/TV: bonevolume/tissue volume *p < 0.05: compared with sham group

The pharmaceutical composition can increase the weight of femur andtibia, bone mineral density, ALP activity, and synthesis of collagen totreat, reduce and ameliorate bone loss.

While the invention has been described by way of example and in terms ofthe preferred embodiments, it is to be understood that the invention isnot limited to the disclosed embodiments. To the contrary, it isintended to cover various modifications and similar arrangements (aswould be apparent to those skilled in the art). Therefore, the scope ofthe appended claims should be accorded the broadest interpretation so asto encompass all such modifications and similar arrangements.

1. A method for preventing, ameliorating and/or treating bone loss,comprising administering to a subject an effective amount of apharmaceutical composition comprising bergapten and a pharmaceuticallyacceptable carrier or excipient.
 2. The method as claimed in claim 1,wherein the bone loss comprises osteoporosis or an osteoporoticfracture.
 3. The method as claimed in claim 1, wherein thepharmaceutical composition increases the alkaline phosphatase (ALP)activity by at least 80.0% in a bone cell, increases the collagensynthesis by at least 100.0% in a bone cell, and/or increases themineralization by at least 105.0% in a bone cell.
 4. The method asclaimed in claim 1, wherein the pharmaceutical composition increasesbone mineral density (BMD) by at least 12.2% and/or increases bonemineral content (BMC) by at least 33.7%.
 5. The method as claimed inclaim 1, wherein the pharmaceutical composition is administrated orallyor by injection.